The overall objective of this project is to design, synthesize and further develop several series of small-molecule drugs that have the common feature of being selectively toxic to hypoxic cells. This approach is based on the fact that hypoxia is much more severe in solid tumors than normal tissue. We will explore several approaches to the development of such drugs, which have the promise of being less toxic than current cytotoxic agents to normal tissue, and therefore having better therapeutic ratios. We will follow up progress made in the current program in defining optimal physicochemical properties (solubility, lipophilicity, reduction potential, radical stability, DNA targeting) of analogs of the hypoxia-activated prodrug tirapazamine now in Phase III clinical trials. In particular, we will prepare and explore the utility of 3-substituted analogs to optimize many of these properties. We will prepare and characterize novel prodrugs of potent alkylating agents, designed for activation by nitroreductase, beta-glucuronidase and beta-galactosidase enzymes isolated from the bacteria Escherischia coli and from Clostridium species. The genes for these enzymes will be delivered selectively to hypoxic tumor cells by the anaerobic bacterium Clostridia sporogenes, in a therapy approach called Clostridia-directed enzyme-prodrug therapy (CDEPT). We will explore the positioning of the cytotoxin and trigger units within the prodrug to optimize prodrug/enzyme interactions, and will determine the appropriate lipophilicities of the prodrugs and their released effectors for optimum extravascular diffusion, in developing prodrugs for this exciting new vector delivery system. We will study compounds that are lethal to cells with upregulated activity of hypoxia-induced transcription factor 1alpha (HIF-1alpha). This is an important component of the pathway by which cells adapt to low-oxygen environments, and drugs that are preferentially cytotoxic in its presence or in the presence of downstream enzymes upregulated by it, are expected to be selectively toxic to hypoxic cells as well as to aerobic cells over-expressing HIF-1alpha. We will evaluate hits in terms of their potential development as therapeutic drugs (e.g., solubility, diffusional properties, ease of synthesis, novelty). We will then prepare small series of analogs of selected lead small molecules, to identify classes suitable for advanced development.